The present invention relates to an assay method, for example, using the pinna reflex, and is more particularly directed to an assay method for determining the potency of a substance, for example, botulinum toxin type A.
Botulinum toxin, for example, botulinum toxin type A, has been used in the treatment of a number of neuromuscular disorders and conditions involving muscular spasm, such as strabismus, blepharospasm, spasmodic torticollis (cervical dystonia), oromandibular dystonia, spasmodic dysphonia (laryngeal dystonia) and the like. The toxin binds rapidly and strongly to presynaptic cholinergic nerve terminals and inhibits the exocytosis of acetylcholine. This results in local paralysis thereby relaxing the muscle afflicted by spasm.
For one example of treating neuromuscular disorders, see U.S. Pat. No. 5,053,005, which suggests treating curvature of the juvenile spine, i.e., scoliosis, with an acetylcholine release inhibitor, preferably botulinum toxin A. For the treatment of strabismus with botulinum toxin type A, see Elston, J. S., et al., British Journal of Ophthalmology, 1985, 69, 718–724 and 891–896. For the treatment of blepharospasm with botulinum toxin type A, see Adenis, J. P., et al., J. Fr. Ophthalmol., 1990, 13 (5) at pages 259–264. For treating spasmodic and oromandibular dystonia torticollis, see Jankovic et al., Neurology, 1987, 37, 616–623. Spasmodic dysphonia has also been treated with botulinum toxin type A. See Blitzer et al., Ann. Otol. Rhino. Laryngol, 1985, 94, 591–594. Lingual dystonia was treated with botulinum toxin type A according to Brin et al., Adv. Neurol. (1987) 50, 599–608. Cohen et al., Neurology (1987) 37 (Suppl. 1), 123–4, discloses the treatment of writer's cramp with botulinum toxin type A.
Botulinum toxin is a generic term embracing the family of toxins produced by the anaerobic bacterium Clostridium botulinum. To date seven immunologically distinct neurotoxins serotype have been identified. These have been given the designations A, B, C1, D, E, F and G. For further information concerning the properties of the various botulinum toxins see, Jankovic and Brin, The New England Journal of Medicine, Vol. 324, No. 17, 1990, pp. 1186–1194, and see, the review by Charles L. Hatheway in Chapter 1 of the book entitled Botulinum Neurotoxin and Tetanus Toxin, L. L. Simpson, Ed., published by Academic Press Inc. of San Diego, Calif., 1989.
Botulinum toxin is obtained commercially by growing cultures of C. botulinum in a fermenter and then harvesting and purifying the fermented mixture in accordance with known techniques. Botulinum toxin type A, the toxin type generally utilized in treating neuromuscular conditions, is currently available commercially from several sources including from Allergan, Inc., Irvine, Calif., under the trade name BOTOX7, and from Porton Products Ltd. UK, under the trade name DYSPORT7.
The active component of the botulinum toxin type A in therapeutic preparations is complexed to the non-toxic component haemagglutinin, and is present in extremely small amounts. The specific activities of different formulations vary because retention of biological potency during the formulation freeze-drying process is not reproducible. This results in different ratios of active to inactive toxin and makes it impossible to express the unit activity of this compound in terms of mass (Hambleton, J Neurol 1992, 239:16–20; Schantz and Johnson, Microbiol Rev 1992, 56:80–99).
At present the biological potency of therapeutic preparations of type A botulinum toxin is expressed in terms of mouse LD50 units. Contrary to general belief, the mouse unit is not a standardized unit. It is well documented that the assay to determine the potency of botulinum toxin type A in mouse LD50 units is prone to significant inter-laboratory variability (Schantz and Kautter, J Ass of Anal Chem 1978, 61:96–99). One study designed to standardize a Botulinum type A toxin assay involved 11 different laboratories (Sesardic et al, Pharacol Toxico 1996, 78:283–288). In this study there was found to be up to a 10-fold difference in results. This variability in mouse LD50 is not unique to assays involving botulinum toxin. In fact, because of the variability of this assay, a number of regulatory agencies have abandoned requiring the routine use of LD50 for toxicity testing for a number of chemicals, solvents, cosmetics and drugs (Pearce et al, Toxicol App Pharm 1994, 128:69–77).
The expanding medical importance of botulinum toxins has increased the need for, and placed a premium on, the precise analysis of biological activity contained in preparations of botulinum toxin type A for both clinical use and laboratory investigation.
It would be advantageous to provide a more precise measurement of toxin activity based on a non-lethal exposure of botulinum toxin type A to rats.
The pinna reflex is a part of the overall acoustic startle response that occurs in certain mammals, including the rat. The characteristic feature of the acoustic startle response in mammals is a pattern of generalized muscular contractions with short latencies. Reflex action starts in the face and neck and spreads down the body, producing a transitory crouch-like gesture. The prominent motor component of the startle response is a flexor contraction.
Response probability and magnitude of the reflex rises with an increase in stimulus intensity. In addition to the response of the body muscles, the whole body startle, several muscles of the head are also involved in the startle reaction. Additionally, a flexion of the pinna occurs in some mammals including rats. This is called the Preyer reflex. Pinna motor reactions caused by intense acoustic stimulation are described as an essential component of the startle reaction with response similar to the whole-body startle. However, this movement of the ears may be a separate reflex.